1. Technical Field
The claimed subject matter relates to testing blood clotting in blood, and more particularly to determining the anti-Factor Xa and/or anti Factor IIa activities of compounds in a blood sample.
2. Background
The determination of heparin establishes an important parameter for the supervision of heparin treatment, which is often administered in the presence of a threat of thrombosis. Heparin forms with antithrombin III (AT III) a complex that inhibits the proteolytic activity of FXa and thrombin. Heparin treatment is often applied in the presence of a threat of thrombosis (e.g. before surgical interventions). Adjustment of heparin concentration is therefore important. If the dose is too low, there is the danger of thrombosis or embolism, which can result in death. Excessively high heparin concentrations, however, result in bleeding. The quantitative analysis of heparin, therefore, is one of the tests most frequently performed in a blood testing laboratory.
In 1973 Yin and co-workers developed the first quantitative assay method for the in vitro measurement of heparin in plasma based on Factor Xa neutralization (J. Lab. Clin. Med. 81:298, 1973). This assay method could detect less than 0.02 units of heparin per milliliter of plasma. However, the assay is cumbersome and time consuming to perform. It is a two-stage assay, and requires much manual manipulation. In the first-stage of the assay, the patient's plasma sample is incubated with the sample plasma, buffer, and a known excess of Factor Xa for a predetermined time period, after which a sub sample from this primary reaction mixture is removed and assayed for residual Factor Xa activity. The latter step constitutes the second-stage of the assay. The residual Factor Xa activity is measured by the addition of the test sample to another test tube, and to it calcium chloride, cephalin in bovine plasma are added separately in timed fashion. Other assays that presently exist are either cumbersome or limited in scope of application. Therefore, a need exists for improved heparin assay methods.